Totally support the statements below. We have had several proteins with A280 absorbance of 0.1 or less (at 1 mg/ml). You _have_ to use Bradford in the Nanodrop or whatnot to measure the concentration.
Before purchasing the Nanodrop we used a Hellma TrayCell and a "normal" UV/Vis instrument. Similar to the Nanodrop, the sample volume in TrayCell is 2-3 ul. Traycell works well at a fraction of the Nanodrop cost, but Nanodrop is a lot more convenient to use for high concentration quick measurements (especially if you need to measure several things in succession), so you get what you pay for. Petr P.S. Expasy's Protparam tool has been around for ages (10-12+ years?). That plus the Nanodrop are two essential and synergetic tools of a protein chemist/crystallographer. On Jun 16, 2011, at 10:31 PM, Edward A. Berry wrote: >> > Bradford is an assay, Nanodrop is a spectrophotometer. > Both the A280 and Bradford methods are strongly dependent on > amino acid composition, so unless you correct A280 for that > as mentioned by Filip, either one is semiquantitative. > Occasionally you come across a protein with no tryptophan > which will have a much lower extinction coefficient. > Try making a 1 g/l solution of gelatin (collagen?) > and see what its A280 is! I noticed recently the > "protparam" tool at http://ca.expasy.org/cgi-bin/protparam > estimates the extinction coefficient given a sequence. > > > > David Briggs wrote: > ~~~ >> >> I wouldn't touch Bradford with a barge-pole. I've found it to be >> wildly inaccurate for certain proteins I've handled, where as the >> OD280 measurements have been fine. >> > One wonders what does "fine" mean, like same as with Biuret or > Kjeldahl nitrogen, or solution made up by weight?
